P2X7 receptor antagonism in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive disorder characterized by the degeneration and subsequent loss of upper and lower motor neurons, resulting in reduced muscle function and paralysis (Mathis et al., 2017). Current therapies are limited to the oral administration of riluzole, which improves survival by only 3 months (Mathis et al., 2017). Thus, new therapies are urgently required to treat this neurodegenerative disorder. Over the past decade, the intercellular signaling pathway involving extracellular adenosine triphosphate (ATP) activation of the P2X7 receptor channel has emerged as a potential therapeutic target in ALS (Volonte et al., 2016). To this end, we evaluated recently whether the P2X7 antagonist, Brilliant Blue G (BBG), could alter disease progression in a murine model of ALS (Bartlett et al., 2017). In this model, mice overexpress a human variant of mutant superoxide dismutase 1 (SOD1) and typically develop clinical signs of ALS from day 70 or later depending on the laboratory, and the transgene copy number, gender and genetic background of the mice (Heiman-Patterson et al., 2005). Mutations in the gene encoding SOD1 (including SOD1) are associated with 15–20% of familial ALS cases in humans, with human SOD1 transgenic mice being one of the most commonly used animal models of this disease (Heiman-Patterson et al., 2005). In our study (Bartlett et al., 2017), intraperitoneal (i.p.) injection of BBG (45.5 mg/kg) into SOD1 mice 3 times per week from 62 days of age (late pre-onset) delayed weight loss and prolonged survival of female, but not male, mice. BBG treatment also delayed weight loss in both genders combined. BBG treatment had no effect on clinical score or motor coordination in either gender, nor did it prevent motor neuron loss, microgliosis, or affect the amount of lumber SOD1 or P2X7 protein at end-stage. Therefore, results from this study demonstrate that P2X7 antagonism with BBG has some therapeutic benefit in ALS, at least in female SOD1 mice. During the course of our pre-clinical trial (Bartlett et al., 2017), two similar studies were published (Cervetto et al., 2013; Apolloni et al., 2014). These studies also reported some therapeutic benefit with BBG treatment in SOD1 mice, but with notable differences to our study (Figure 1). In the first of these studies (Cervetto et al., 2013), injection of BBG (45.5 mg/kg i.p.) every 2 days from day 90 (onset) improved motor coordination in mice of either gender, with greater improvement in male than female mice. BBG treatment also delayed weight loss in male and both genders combined, but not in female mice. BBG treatment did not alter survival. In the second of these studies (Apolloni et al., 2014), injection of BBG (50 mg/kg i.p.) 3 times per week from day 100 (late pre-onset) improved motor coordination, but did not alter disease onset or survival, with no differences observed between genders. Injection of BBG (50 mg/kg i.p.) 3 times per week from day 135 (onset) had no effect on motor coordination, disease onset or survival (Apolloni et al., 2014). SOD1 mice were also injected with a higher dose of BBG (250 mg/kg i.p.) 3 times per week from either day 40 (asymptomatic), day 70 (pre-onset) or day 100 (late pre-onset) (Apolloni et al., 2014). BBG treatment from day 40 or 70 did not alter any observed disease parameter, but BBG treatment from day 100 delayed disease onset, and improved clinical score and motor coordination, but did not delay weight loss or prolong survival. BBG treatment from day 100 also increased motor neuron survival, and reduced microgliosis and pro-inflammatory markers (nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and interleukin-1β) at end-stage. As with treatment using BBG at 50 mg/kg, no differences were observed between genders with this compound at 250 mg/kg. The effect of BBG treatment on the survival of female SOD1 mice in our study (Bartlett et al., 2017) paralleled the prolonged survival observed in female, but not male, heterozygous and homozygous P2X7 knockout (P2X7)/SOD1 mice compared to P2X7 wild-type (P2X7)/SOD1 mice (Apolloni et al., 2013). In contrast, genetic ablation of P2X7 anticipated clinical onset, worsened disease progression and increased motor neuron loss, microgliosis, astrogliosis and pro-inflammatory markers (NADPH oxidase and inducible nitric oxide synthase) towards or at end-stage (Apolloni et al., 2013). Consistent with this, BBG treatment in our study caused an increase in pro-inflammatory marker serum monocyte chemoattractant protein-1 in end-stage SOD1 mice (Bartlett et al., 2017), although group sizes were insufficiently powered to detect a significant difference. Together these two studies demonstrate a dual role for P2X7 activation in this model of ALS (at least in female SOD1 mice) by promoting disease progression but limiting aspects of neuroinflammation. Moreover, it may be of value to compare the effects of BBG treatment in P2X7/SOD1 and P2X7/SOD1 mice (which to the best of our knowledge has not been reported) to confirm P2X7 antagonism by BBG in SOD1 mice expressing functional P2X7. Collectively the four studies discussed above (Apolloni et al., 2013, 2014; Cervetto et al., 2013; Bartlett et al., 2017) reveal a complex role for P2X7 in the SOD1 murine model of ALS and by extension ALS in humans. The various and contrasting gender differences observed with P2X7 antagonism or genetic ablation in these studies remain unexplained, but it is well known that disease progression in this model of ALS is gender dependent (Heiman-Patterson et al., 2005). It is noteworthy, that disease progression accelerates in ovariectomized SOD1 mice and that this effect can be reversed by administration of 17β-oestradiol (Choi et al., 2008), a compound which can also inhibit P2X7 activation (Cario-Toumaniantz et al., 1998). Whilst, these